WO2023155352A1 - Test method and test kit for lipoprotein cholesterol - Google Patents
Test method and test kit for lipoprotein cholesterolDownload PDFInfo
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- WO2023155352A1 WO2023155352A1PCT/CN2022/100428CN2022100428WWO2023155352A1WO 2023155352 A1WO2023155352 A1WO 2023155352A1CN 2022100428 WCN2022100428 WCN 2022100428WWO 2023155352 A1WO2023155352 A1WO 2023155352A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
- C12Q2326/32—3-Methyl-2-benzothiazolinone hydrazone hydrochloride hydrate, i.e. MBTH
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
- G01N2333/918—Carboxylic ester hydrolases (3.1.1)
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
Definitions
- the present inventionrelates to a method and a reagent for measuring lipoprotein cholesterol, in particular to a method and a reagent for detecting lipoprotein cholesterol related to the diagnosis of arteriosclerosis.
- LDLLow-density lipoprotein
- Examples of conventional methods for measuring small, dense LDLinclude ultracentrifugation, electrophoresis, and methods using high performance liquid chromatography. Since these methods require expensive equipment and a lot of measurement time, they are not convenient.
- An example of a method for measuring small, dense LDL using an automatic analyzeris the following method (see Japanese Patent Publication No. 2003-28882A), which involves suspending or dissolving small-particle LDL using a difference in ionic strength, and then using a difference in absorbance to Small particle LDL was measured.
- the difference in absorbanceis based on turbidimetry according to the method described above. Therefore, cholesterol in small and dense LDL cannot be determined, so the specificity and accuracy are insufficient.
- a methodis known (see International Patent Publication WO2004/053500) which involves measuring the concentration of ⁇ in small, dense LDL by using a combination of a separating agent comprising polyanions and divalent cations and a reagent suitable for an automatic analyzer.
- a separating agentcomprising polyanions and divalent cations
- a reagent suitable for an automatic analyzercholesterol or triglycerides.
- This methodcan measure lipid components in small and dense LDL more conveniently than ultracentrifugation or electrophoresis. But this method requires pretreatment of the sample and a step of separating LDL into small dense LDL and LDL other than such LDL.
- the inventionrelates to a method for the fractional determination of lipoprotein cholesterol suitable for an automatic analyzer and reagents for such determination. In the case of clinical test results, it meets the linearity requirements of the reagent, with high accuracy and good stability.
- compositioncomprises:
- the compositionalso comprises a preservative.
- the preservativeis one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
- the present inventionalso provides a composition, characterized in that the composition comprises:
- the preservativeis one or more of sodium azide, Florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
- the aforementioned compositionis in an acidic environment.
- the pH valueis 0.5-4.5, more preferably, the pH value is 1-4.05.
- the above compositionfurther comprises one or more selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
- the composition described abovealso comprises a surfactant.
- the surfactantis selected from polyoxyethylene derivatives, preferably polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether (EMULGEN707, EMULGEN709), polyoxyethylene alkylphenyl ether, more preferably, Polyethylene glycol octylphenyl ether (Triton X-100), polyoxyethylene nonylphenyl ether (EMULGEN909).
- the concentration of the 3-methylbenzothiazolone hydrazone or its saltis 0.1-1.0 g/L. More preferably, the concentration of the 3-methylbenzothiazolone hydrazone or its salt is 0.1-0.4 g/L.
- the concentration of peroxidaseis 2000-4000U/L.
- the concentration of the surfactantis 20-35g/L.
- the present inventionalso provides a kit, which is characterized in that the kit includes at least the reagent composition (ii), and the reagent composition (ii) includes the above-mentioned composition.
- the kitfurther comprises a reagent composition (i), and the reagent composition (i) is used for effectively shielding interferences other than the lipoprotein to be tested.
- the reagent composition (i)comprises phospholipase, preferably, at least one of phospholipase A2, phospholipase C, phospholipase D, lysophospholipase (LYPL) and sphingomyelinase; the concentration range of the preferred phospholipase 0.1U/mL-100U/mL.
- effective shielding in the present inventionis to act on the interfering components through physical or chemical means, so that the interfering components will not have a substantial impact on the subsequent detection steps, and effective shielding does not require that the interfering components be completely removed or excluded from the system outside.
- the reagent composition (i)also includes a nonionic surfactant, preferably a nonionic surfactant of HLB 13-15, more preferably, a polyoxyethylene derivative or polyoxyethylene distyrylphenyl Ether, for example, EMULGEN B-66, EMULGEN A-90;
- a nonionic surfactantpreferably a nonionic surfactant of HLB 13-15, more preferably, a polyoxyethylene derivative or polyoxyethylene distyrylphenyl Ether, for example, EMULGEN B-66, EMULGEN A-90;
- concentration of said nonionic surfactantis 1.5-2.0g/L.
- the reagent composition (i)further comprises cholesterol esterase, cholesterol oxidase and hydrogen donor.
- the reagent composition (i)also includes inorganic salt ions, selected from one or more of magnesium chloride, EDTA 2Na, EDTA 2K, sodium glutamate, sodium chloride or potassium chloride .
- the reagent composition (i)further includes a stabilizer selected from one or more of BSA, sucrose or trehalose.
- the hydrogen donoris an aniline derivative, preferably TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline).
- the present inventionalso provides a preparation method of the above composition and/or kit.
- the present inventionalso provides the application of the above composition and/or kit.
- composition and/or kitare used in the preparation of products for detecting lipoprotein cholesterol.
- the productincludes a reagent or a test kit.
- the detection of lipoprotein cholesterolincludes detection of low-density lipoprotein cholesterol, small dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, remnant-like lipoprotein cholesterol or total lipoprotein cholesterol.
- compositions and/or kitare also used in the preparation of products for diagnosing cardiovascular related diseases.
- the productincludes a reagent or a test kit.
- cardiovascular-related diseasessuch as dyslipidemia, especially hyperlipidemia, hypercholesterolemia, atherosclerosis and other diseases, including low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein Detection of protein cholesterol, remnant lipoprotein cholesterol, or total lipoprotein cholesterol.
- the present inventionalso provides a method for improving the linearity or accuracy of lipoprotein cholesterol detection, which is characterized by using the above composition and/or kit.
- the present inventionalso provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or its salt, which is characterized by containing a preservative.
- the preservativeis one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
- the 3-methylbenzothiazolone hydrazone or its saltis preferably in an acidic environment.
- the pHis 1.00-4.05. More preferably, it contains one or more acids or buffer systems selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
- the present inventionalso provides a method for quantitatively determining lipoprotein cholesterol in a sample, which is characterized by using the above composition and/or the reagent composition (ii) in the kit.
- the methodincludes the following steps: adding the reagent composition (ii) in the above composition and/or kit to detect target lipoprotein cholesterol. Further, the method includes the following steps:
- italso includes adding cholesterol esterase, cholesterol oxidase and chromogen (hydrogen donor) in step (1) or (2).
- the reagent composition (i)comprising adding the above kit in step (1).
- the kitis used for the detection of low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, residual particle-like lipoprotein cholesterol or total lipoprotein cholesterol.
- the step (1)can be further subdivided into multiple steps to effectively shield different interference components.
- the present inventionprovides a method for measuring lipoprotein cholesterol suitable for an automatic analyzer and a reagent for such determination.
- the method and the reagentcan be carried out without pretreatment of the sample, and have good specificity, rapidity and It is convenient for analysis, and at the same time, it also ensures that the determination reagent can meet the linearity requirements of the reagent without affecting the clinical test results, and has the characteristics of high accuracy and good stability.
- the present inventionalso provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or a salt thereof, a related composition and a kit.
- Figure 1shows the changes in the reactivity of 3-methylbenzothiazolone hydrazone after acceleration with different buffer types.
- Fig. 2is a random sample correlation analysis between the reagent of Example 10 and the commercially available reagent (Jiuqiang 20-0630).
- Fig. 3is a random sample correlation analysis between the reagent of Example 11 and the commercially available reagent (Jiuqiang 20-0630).
- Fig. 4is a random sample correlation analysis between the reagent of Example 12 and the commercially available reagent (Jiuqiang 20-0630).
- the kitis divided into two reagent compositions, R1 and R2, and the specific formula is as follows:
- Embodiment 2Measuring method
- the volume ratio of the reagent R1 to the reagent R2is 3:150:50.
- the correlation coefficient rshould be ⁇ 0.990.
- the absolute deviation of linearityshould not exceed ⁇ 20 mg/L;
- the linear relative deviationshould not exceed ⁇ 10%.
- Reagent blankUse purified water or a designated blank solution to add reagents as a sample test, which is an effective indicator for judging the quality of reagents.
- Analytical SensitivityThe slope of the calibration curve.
- LinearityThe ability of a measurement result to be directly proportional to the value measured in the sample.
- Linear rangeThe test system can accept the linear range, that is, the nonlinear error is less than the specified error within this range.
- Interferencerefers to the clinically significant deviation of the concentration of the analyte due to the influence of sample characteristics or other components in clinical chemistry.
- Interference standardthe maximum permissible interference deviation between the concentration of the measured substance and the true value, which may affect the doctor's
- Anti-interferenceDetermine the interfering properties of interfering substances.
- the relative deviation of linearityshould not exceed ⁇ 10%.
- the low-end deviationis large, the linearity is poor, and the external quality assessment deviation is large, which cannot meet the requirements of accurate detection.
- Embodiment 3color source substance concentration screening
- reagents containing different color source substanceswherein TOOS is used as the color source material, and the concentrations are: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2mM.
- concentrationsare: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2mM.
- the correlation coefficient r of the assay kitmeets the requirements, but from Table 2, it can be seen that the low-end deviation of the reagent kit in Table 1 is relatively large, and the linearity is poor.
- the present inventionadjusts the concentration of the color source substance , when the concentration is 0.4mM-3.2mM, it does not conform to the range of 40-200mg/L, and the absolute deviation of linearity should not exceed ⁇ 20mg/L; Within the interval, the linear relative deviation should not exceed ⁇ 10%. It can be seen that the linearity of the kit reagents cannot be optimized by adjusting the concentration of the color source substance.
- the linearity of the reagents in the kitis poor and does not meet the linearity standard of the reagents.
- the linearity of the reagents in the kitcannot be optimized by adjusting the concentration of the color source substance.
- Embodiment 4Surfactant concentration screening
- kits components and contents in Table 1prepare reagents containing different surfactants, wherein polyoxyethylene distyrenated phenyl ether (A90) is used as the surfactant, and the concentrations are: 0.35g/L, 0.7 g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8g/L.
- concentrationsare: 0.35g/L, 0.7 g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8g/L.
- the linearity of the reagents in the kitis poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of the surfactant.
- the linearity of the reagents in the kitis poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of 4-aminoantipyrine.
- Triton-100Prepare reagents containing different concentrations of Triton-100, the concentrations of Triton-100 are: 5g/L, 15g/L, 25g/L, 35g/L, 50g/L. Reagents with different concentrations of Triton-100 were used for linearity verification, and the linearity verification method was the same as above.
- kit reagents in the prior arthave poor linearity and do not meet the linearity standard of the reagents, and the linearity of the kit reagents cannot be optimized by adjusting the concentration of Triton-100.
- Embodiment 7replaces the reagent of 4-aminoantipyrine in embodiment 1 with 3-methylbenzothiazolone hydrazone
- Accelerated calibration deviation calculation formula(37°C calibration response -4°C calibration response)/4°C calibration response.
- the present inventorsunexpectedly found that the linearity of the reagent is optimized after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone substrate, which meets the requirements of the linearity standard for the reagent.
- reagents containing 3-methylbenzothiazolone hydrazone and PC-300 in R2but with different buffer types, in which the concentration of 3-methylbenzothiazolone hydrazone is 0.4mol/L, and different types of buffers include: citric acid Buffer, Succinic Acid, Boric Acid, Tartaric Acid, Glycine, Hydrochloric Acid, Sulfuric Acid, Acetic Acid.
- the correlation coefficient r of the assay reagentis in compliance with the regulations, as can be seen from Table 8 and Figure 1: in the range of pH 1.00-4.05, citric acid, succinic acid, boric acid, tartaric acid, glycine,
- the acidic environmentsuch as hydrochloric acid, acetic acid and its buffer system has a synergistic effect with the preservative PC-300, the stability of the reagent is significantly improved, and unexpected technical effects have been achieved.
- the reagents using 4-aminoantipyrine in the prior artall do not meet the linearity requirements of the reagents, even by adjusting the concentration of the color source substance, the concentration of the surfactant, the concentration of 4-aminoantipyrine, and the concentration of Triton-100 After that, the linearity cannot be optimized, and the linearity cannot meet the requirements of reagent accuracy.
- the linearity of the reagentis improved after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone, which meets the requirement of reagent accuracy, but the inventor finds that the stability of the system is not high.
- PC-300was selected as a preservative, which has the function of stabilizing the reagent system and has met the requirements of performance optimization. Further, by adjusting the pH value to the range of 1.00-4.05, it can produce a synergistic effect with PC-300, greatly improve the stability of the system, and achieve unexpected technical effects.
- the correlation coefficient r using the 3-methylbenzothiazolone assay reagent of the present inventionis in compliance with the regulations, and the absolute deviation and relative deviation in the low value area are very small, fully meeting the linearity requirement.
- Reagent R1Reagent R2: Buffer (PIPES) 100mmol/L PBS-citric acid 100mmol/L pH 2.74 Peroxidase 500KU/L Peroxidase 10000KU/L Cholesterol lipase 600KU/L 3-Methylbenzothiazolone hydrazone 0.8mol/L Cholesterol oxidase 500KU/L Emulgen 909 25g/L
- PBS-citric acid100mmol/L pH 2.74
- the solventis purified water Sodium azide 0.9g/L the
- the solventis purified water
- Embodiment 13Reagent of the present application and commercially available product correlation verification
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Abstract
Description
Translated fromChinese本发明涉及用于测定脂蛋白中胆固醇的方法和试剂，尤其涉及动脉硬化的诊断相关的脂蛋白胆固醇检测的方法和试剂。The present invention relates to a method and a reagent for measuring lipoprotein cholesterol, in particular to a method and a reagent for detecting lipoprotein cholesterol related to the diagnosis of arteriosclerosis.
胆固醇是细胞的一种重要组分，也是临床上重要的组分，因为过高水平的胆固醇导致在内皮下间隙中的巨噬细胞摄取胆固醇后使巨噬细胞转化为泡沫细胞(foam cell)并继而导致动脉硬化的原发病灶的发展。低密度脂蛋白(LDL)在血液的胆固醇运输中起主要作用并且是动脉硬化的危险因素。已知小而密LDL，其在LDL中粒度特别小并且与标准LDL相比密度更高，具有多于正常LDL数倍水平的致动脉粥样化的能力。小而密LDL的增加是动脉硬化的主要危险因素之一。因此在临床上进行此类小而密LDL的分级测定(fractional measurement)是非常重要的。Cholesterol is an important component of cells and is clinically important because excessive levels of cholesterol lead to cholesterol uptake by macrophages in the subendothelial space that convert macrophages into foam cells and This in turn leads to the development of primary lesions of atherosclerosis. Low-density lipoprotein (LDL) plays a major role in cholesterol transport in the blood and is a risk factor for arteriosclerosis. Small dense LDL, which is particularly small in size in LDL and denser than standard LDL, is known to have an atherogenic potential several times higher than normal LDL levels. The increase of small and dense LDL is one of the main risk factors of arteriosclerosis. Therefore, it is very important to conduct fractional measurement of such small and dense LDL clinically.
用于测定小而密LDL的常规方法的实例包括超速离心法、电泳法以及使用高效液相色谱的方法。由于这些方法需要昂贵的设备和大量测量时间，因此并不方便。Examples of conventional methods for measuring small, dense LDL include ultracentrifugation, electrophoresis, and methods using high performance liquid chromatography. Since these methods require expensive equipment and a lot of measurement time, they are not convenient.
一种利用自动分析仪测量小而密LDL的方法的实例是以下方法(参见日本专利公布第2003-28882A号)，该方法涉及利用离子强度差异将小颗粒LDL悬浮或溶解，然后利用吸光度差异对小颗粒LDL进行测定。然而，吸光度差异依照上述方法是基于浊度测定的。因此不能测定小而密LDL中胆固醇，因而特异性和准确度不足。An example of a method for measuring small, dense LDL using an automatic analyzer is the following method (see Japanese Patent Publication No. 2003-28882A), which involves suspending or dissolving small-particle LDL using a difference in ionic strength, and then using a difference in absorbance to Small particle LDL was measured. However, the difference in absorbance is based on turbidimetry according to the method described above. Therefore, cholesterol in small and dense LDL cannot be determined, so the specificity and accuracy are insufficient.
此外，已知一种方法(参见国际专利公布WO2004/053500)，该方法涉及通过使用包含聚阴离子和二价阳离子的分离剂与适合于自动分析仪的试剂的组合来测量小而密LDL中的胆固醇或三酰甘油。该方法能够比超速离心法或电泳法更方便地测量小而密LDL中的脂质组分。但是该方法需要预处理试样并需要将LDL分离成小而密LDL和除这种LDL之外的LDL的步骤。In addition, a method is known (see International Patent Publication WO2004/053500) which involves measuring the concentration of β in small, dense LDL by using a combination of a separating agent comprising polyanions and divalent cations and a reagent suitable for an automatic analyzer. cholesterol or triglycerides. This method can measure lipid components in small and dense LDL more conveniently than ultracentrifugation or electrophoresis. But this method requires pretreatment of the sample and a step of separating LDL into small dense LDL and LDL other than such LDL.
现有技术中公开了一种方法(参见国际专利公布WO2007026829A1)，该方法涉及通过特定表面活性剂清除除小而密LDL之外的脂蛋白中的胆固醇，然后测定剩余的脂蛋白中的胆固醇的量。该方法能够在无需预处理试样下进行具有快速且方便的分析。然而，经实验发现，该方法测定试剂低端偏差较大，线性较差，室间质评偏差大，无法达到准确检测的要求。A method is disclosed in the prior art (see International Patent Publication WO2007026829A1), which involves removing cholesterol from lipoproteins other than small, dense LDL by specific surfactants, and then measuring the cholesterol in the remaining lipoproteins. quantity. This method enables rapid and convenient analysis without pretreatment of samples. However, it was found through experiments that the low-end deviation of the reagents determined by this method was large, the linearity was poor, and the external quality assessment deviation was large, which could not meet the requirements of accurate detection.
发明内容Contents of the invention
本发明涉及一种适用于自动分析仪的分级测定脂蛋白胆固醇的方法以及用于此类测定的试剂，所述方法和试剂能够在无需预处理试样下进行，同时保证测定试剂能在不影响临床检测结果的情况下，满足试剂线性要求，准确度高并且稳定性好。The invention relates to a method for the fractional determination of lipoprotein cholesterol suitable for an automatic analyzer and reagents for such determination. In the case of clinical test results, it meets the linearity requirements of the reagent, with high accuracy and good stability.
本发明通过如下技术方案来实现：The present invention is realized through the following technical solutions:
本发明提供了一种组合物，其特征在于，所述组合物包含：The present invention provides a kind of composition, it is characterized in that, described composition comprises:
3-甲基苯并噻唑酮腙或其盐；和3-Methylbenzothiazolone hydrazone or a salt thereof; and
过氧化物酶。peroxidase.
根据本发明，所述组合物还包含防腐剂。优选地，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。According to the invention, the composition also comprises a preservative. Preferably, the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
本发明还提供了一种组合物，其特征在于，所述组合物包含：The present invention also provides a composition, characterized in that the composition comprises:
3-甲基苯并噻唑酮腙或其盐；和3-Methylbenzothiazolone hydrazone or a salt thereof; and
防腐剂。preservative.
根据本发明，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。According to the present invention, the preservative is one or more of sodium azide, Florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
根据本发明，上述组合物在酸性环境中。According to the invention, the aforementioned composition is in an acidic environment.
根据本发明，优选地，pH值为0.5-4.5，更优选地，pH值为1-4.05。According to the present invention, preferably, the pH value is 0.5-4.5, more preferably, the pH value is 1-4.05.
根据本发明，更优选地，上述组合物还包含选自柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系中的一种或多种。According to the present invention, more preferably, the above composition further comprises one or more selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
根据本发明，上述组合物还包含表面活性剂。其中，所述表面活性剂选自聚氧乙烯衍生物，优选聚氧乙烯-聚氧丙烯共聚物、聚氧乙烯烷基醚(EMULGEN707，EMULGEN709)、聚氧乙烯烷基苯基醚，更优选，聚乙二醇辛基苯基醚(Triton X-100)，聚氧乙烯壬基苯基醚(EMULGEN909)。According to the invention, the composition described above also comprises a surfactant. Wherein, the surfactant is selected from polyoxyethylene derivatives, preferably polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene alkyl ether (EMULGEN707, EMULGEN709), polyoxyethylene alkylphenyl ether, more preferably, Polyethylene glycol octylphenyl ether (Triton X-100), polyoxyethylene nonylphenyl ether (EMULGEN909).
根据本发明，所述3-甲基苯并噻唑酮腙或其盐的浓度为0.1-1.0g/L。更优选，所述3-甲基苯并噻唑酮腙或其盐的浓度为0.1-0.4g/L。过氧化物酶的浓度为2000-4000U/L。所述表面活性剂的浓度为20-35g/L。According to the present invention, the concentration of the 3-methylbenzothiazolone hydrazone or its salt is 0.1-1.0 g/L. More preferably, the concentration of the 3-methylbenzothiazolone hydrazone or its salt is 0.1-0.4 g/L. The concentration of peroxidase is 2000-4000U/L. The concentration of the surfactant is 20-35g/L.
本发明还提供了一种试剂盒，其特征在于，所述试剂盒至少包含试剂组合物(ii)，所述试剂组合物(ii)包含上述组合物。The present invention also provides a kit, which is characterized in that the kit includes at least the reagent composition (ii), and the reagent composition (ii) includes the above-mentioned composition.
根据本发明，所述试剂盒还包含试剂组合物(i)，所述试剂组合物(i)用于有效屏蔽除待测脂蛋白之外的干扰。优选地，所述试剂组合物(i)包含磷脂酶，优选，磷脂酶A2、磷脂酶C、磷脂酶D、溶血磷脂酶(LYPL)和鞘磷脂酶的至少一种；优选磷脂酶的浓度范围为0.1U/mL-100U/mL。According to the present invention, the kit further comprises a reagent composition (i), and the reagent composition (i) is used for effectively shielding interferences other than the lipoprotein to be tested. Preferably, the reagent composition (i) comprises phospholipase, preferably, at least one of phospholipase A2, phospholipase C, phospholipase D, lysophospholipase (LYPL) and sphingomyelinase; the concentration range of the preferred phospholipase 0.1U/mL-100U/mL.
本发明所述有效屏蔽的定义为，通过物理或化学的手段作用于干扰成分，使干扰成分对后续的检测步骤不会产生实质影响，有效屏蔽并不要求将干扰成分完全清除或排除于体系之外。The definition of effective shielding in the present invention is to act on the interfering components through physical or chemical means, so that the interfering components will not have a substantial impact on the subsequent detection steps, and effective shielding does not require that the interfering components be completely removed or excluded from the system outside.
根据本发明，所述试剂组合物(i)还包括非离子型表面活性剂，优选HLB 13-15的非离子表面活性剂，更优选，聚氧乙烯衍生物或聚氧乙烯二苯乙烯苯基醚，例如，EMULGEN B-66,EMULGEN A-90；优选所述非离子表面活性剂的浓度为1.5-2.0g/L。According to the present invention, the reagent composition (i) also includes a nonionic surfactant, preferably a nonionic surfactant of HLB 13-15, more preferably, a polyoxyethylene derivative or polyoxyethylene distyrylphenyl Ether, for example, EMULGEN B-66, EMULGEN A-90; Preferably the concentration of said nonionic surfactant is 1.5-2.0g/L.
根据本发明，所述试剂组合物(i)还包括胆固醇酯酶、胆固醇氧化酶和氢供体。According to the present invention, the reagent composition (i) further comprises cholesterol esterase, cholesterol oxidase and hydrogen donor.
根据本发明，所述试剂组合物(i)还包括无机盐离子，选自：氯化镁、EDTA·2Na、EDTA·2K、谷氨酸钠、氯化钠或氯化钾中的一种或多种。According to the present invention, the reagent composition (i) also includes inorganic salt ions, selected from one or more of magnesium chloride, EDTA 2Na, EDTA 2K, sodium glutamate, sodium chloride or potassium chloride .
根据本发明，所述试剂组合物(i)还包括稳定剂，选自：BSA、蔗糖或海藻糖中的一种或多种。According to the present invention, the reagent composition (i) further includes a stabilizer selected from one or more of BSA, sucrose or trehalose.
根据本发明，所述氢供体为苯胺衍生物，优选TOOS(N-乙基-N-(2-羟基-3-磺基丙基)-3-甲基苯胺)。According to the invention, the hydrogen donor is an aniline derivative, preferably TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline).
本发明还提供了上述组合物和/或试剂盒的制备方法。The present invention also provides a preparation method of the above composition and/or kit.
本发明还提供了上述组合物和/或试剂盒的应用。The present invention also provides the application of the above composition and/or kit.
根据本发明，所述组合物和/或试剂盒用于制备检测脂蛋白胆固醇的产品中的应用。According to the present invention, the composition and/or kit are used in the preparation of products for detecting lipoprotein cholesterol.
其中，所述产品包括试剂或试剂盒。所述检测脂蛋白胆固醇包括低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。Wherein, the product includes a reagent or a test kit. The detection of lipoprotein cholesterol includes detection of low-density lipoprotein cholesterol, small dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, remnant-like lipoprotein cholesterol or total lipoprotein cholesterol.
根据本发明，上述组合物和/或试剂盒还用于制备诊断心血管相关疾病的产品中的应用。According to the present invention, the above composition and/or kit are also used in the preparation of products for diagnosing cardiovascular related diseases.
其中，所述产品包括试剂或试剂盒。所述诊断心血管相关疾病，例如血脂异常，尤其是高脂血症、高胆固醇血症、动脉粥样硬化等疾病，包括低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。Wherein, the product includes a reagent or a test kit. The diagnosis of cardiovascular-related diseases, such as dyslipidemia, especially hyperlipidemia, hypercholesterolemia, atherosclerosis and other diseases, including low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein Detection of protein cholesterol, remnant lipoprotein cholesterol, or total lipoprotein cholesterol.
本发明还提供了一种提高脂蛋白胆固醇检测线性或准确性的方法，其特征在于，使用上述组合物和/或试剂盒。The present invention also provides a method for improving the linearity or accuracy of lipoprotein cholesterol detection, which is characterized by using the above composition and/or kit.
本发明还提供了一种提高3-甲基苯并噻唑酮腙或其盐稳定性的方法，其特征在于，含有防腐剂。其中，所述防腐剂为叠氮钠、氟苯尼考或PC-300中的一种或多种。更优选地，所述防腐剂为PC-300和氟苯尼考。The present invention also provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or its salt, which is characterized by containing a preservative. Wherein, the preservative is one or more of sodium azide, florfenicol or PC-300. More preferably, the preservatives are PC-300 and Florfenicol.
根据本发明，所述3-甲基苯并噻唑酮腙或其盐优选在酸性环境中。优选地，pH值为1.00-4.05。更优选地，包含选自柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系中的一种或多种酸或缓冲体系。According to the present invention, the 3-methylbenzothiazolone hydrazone or its salt is preferably in an acidic environment. Preferably, the pH is 1.00-4.05. More preferably, it contains one or more acids or buffer systems selected from citric acid, succinic acid, boric acid, tartaric acid, glycine, hydrochloric acid, acetic acid and buffer systems thereof.
本发明还提供了一种用于定量测定样品中脂蛋白胆固醇的方法，其特征在于使用上述组合物和/或试剂盒中的试剂组合物(ii)。The present invention also provides a method for quantitatively determining lipoprotein cholesterol in a sample, which is characterized by using the above composition and/or the reagent composition (ii) in the kit.
根据本发明，该方法包括以下步骤：加入上述组合物和/或试剂盒中的试剂组合物(ii)对目标脂蛋白胆固醇进行检测。进一步，该方法包括以下步骤：According to the present invention, the method includes the following steps: adding the reagent composition (ii) in the above composition and/or kit to detect target lipoprotein cholesterol. Further, the method includes the following steps:
(1)有效屏蔽除待测脂蛋白之外的干扰成分；(1) Effectively shield the interfering components other than the lipoprotein to be tested;
(2)加入上述组合物和/或试剂盒中的试剂组合物(ii)对目标脂蛋白胆固醇进行检测。(2) adding the above composition and/or the reagent composition (ii) in the kit to detect the target lipoprotein cholesterol.
根据本发明，还包括在步骤(1)或(2)中加入胆固醇酯酶、胆固醇氧化酶和色原(氢供体)。According to the present invention, it also includes adding cholesterol esterase, cholesterol oxidase and chromogen (hydrogen donor) in step (1) or (2).
根据本发明，包括在步骤(1)中加入上述试剂盒的试剂组合物(i)。According to the present invention, the reagent composition (i) comprising adding the above kit in step (1).
根据本发明，所述试剂盒用于低密度脂蛋白胆固醇、小而密低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、残粒样脂蛋白胆固醇或总脂蛋白胆固醇的检测。According to the present invention, the kit is used for the detection of low-density lipoprotein cholesterol, small and dense low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, residual particle-like lipoprotein cholesterol or total lipoprotein cholesterol.
根据本发明，所述步骤(1)可以进一步细分为多个步骤对不同的干扰成分进行有效屏蔽。According to the present invention, the step (1) can be further subdivided into multiple steps to effectively shield different interference components.
本发明的效果Effect of the present invention
本发明提供了一种适用于自动分析仪的测定脂蛋白胆固醇的方法以及用于此类测定的试剂，所述方法和试剂能够在无需预处理试样下进行，具有良好特异性的、快速且方便的分析，同时还保证测定试剂能在不影响临床检测结果的情况下，满足试剂线性要求，准确度高并且稳定性好的特点。此外，本发明还提供了一种提高3-甲基苯并噻唑酮腙或其盐稳定性的方法以及相关的组合物和试剂盒。The present invention provides a method for measuring lipoprotein cholesterol suitable for an automatic analyzer and a reagent for such determination. The method and the reagent can be carried out without pretreatment of the sample, and have good specificity, rapidity and It is convenient for analysis, and at the same time, it also ensures that the determination reagent can meet the linearity requirements of the reagent without affecting the clinical test results, and has the characteristics of high accuracy and good stability. In addition, the present invention also provides a method for improving the stability of 3-methylbenzothiazolone hydrazone or a salt thereof, a related composition and a kit.
图1为不同缓冲类型加速后3-甲基苯并噻唑酮腙反应度变化。Figure 1 shows the changes in the reactivity of 3-methylbenzothiazolone hydrazone after acceleration with different buffer types.
图2为实施例10的试剂与市售试剂(九强20-0630)随机样本相关性分析。Fig. 2 is a random sample correlation analysis between the reagent of Example 10 and the commercially available reagent (Jiuqiang 20-0630).
图3为实施例11的试剂与市售试剂(九强20-0630)随机样本相关性分析。Fig. 3 is a random sample correlation analysis between the reagent of Example 11 and the commercially available reagent (Jiuqiang 20-0630).
图4为实施例12的试剂与市售试剂(九强20-0630)随机样本相关性分析。Fig. 4 is a random sample correlation analysis between the reagent of Example 12 and the commercially available reagent (Jiuqiang 20-0630).
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。The technical solutions of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
实施例1试剂盒的制备The preparation of embodiment 1 kit
以现有技术中小而密低密度脂蛋白胆固醇检测试剂盒为例，试剂盒分为R1和R2两种试剂组合物，具体配方如下：Taking the small and dense low-density lipoprotein cholesterol detection kit in the prior art as an example, the kit is divided into two reagent compositions, R1 and R2, and the specific formula is as follows:
表1试剂盒的组分Table 1 Components of the kit
实施例2测定方法Embodiment 2 Measuring method
(1)将待测的血清样本与试剂R1混匀，37℃恒温5min，测定吸光度A1，再加入试剂R2，混匀，37℃恒温5min，测定吸光度A2，计算ΔA＝A2-A1(1) Mix the serum sample to be tested with reagent R1, keep the temperature at 37°C for 5 minutes, measure the absorbance A1 , then add reagent R2, mix well, keep the temperature at 37°C for 5 minutes, measure the absorbance A2 , calculate ΔA=A2 -A1
(2)用全自动生化分析仪(日立7180)测定反应后的吸光度差值(主波长546nm，副 波长660nm，仪器读点16-34)；(2) Measure the absorbance difference after the reaction with a fully automatic biochemical analyzer (Hitachi 7180) (main wavelength 546nm, secondary wavelength 660nm, instrument reading point 16-34);
(3)根据吸光度变化值计算出样本中sdLDL-C的浓度。(3) Calculate the concentration of sdLDL-C in the sample according to the change value of absorbance.
作为优选，步骤(1)中所述的血清样本，试剂R1与试剂R2的体积比为3:150:50。Preferably, for the serum sample described in step (1), the volume ratio of the reagent R1 to the reagent R2 is 3:150:50.
(4)线性实验方法：(4) Linear experimental method:
用接近线性范围上限的高浓度样品和接近线性范围下限的低浓度样品，混合成7个稀释浓度(稀释倍数见下表)，每个浓度测试3次取平均值，取其测定浓度值(即均值yi)与对应的理论浓度值或稀释倍数(xi)进行回归分析，求出线性回归方程(y＝Ax+B)，其中A是斜率，B是截距，计算线性回归相关系数r，结果应符合要求。Use high-concentration samples close to the upper limit of the linear range and low-concentration samples close to the lower limit of the linear range, and mix them into 7 dilution concentrations (see the table below for the dilution factor). Mean value yi ) and corresponding theoretical concentration value or dilution factor (xi) carry out regression analysis, obtain linear regression equation (y=Ax+B), wherein A is slope, B is intercept, calculates linear regression correlation coefficient r, The result should meet the requirements.
将自变量(xi，0值除外)代入线性回归方程，计算因变量(yi)的估计值(y′)，以及yi与y′的绝对偏差和相对偏差，结果应符合要求。Substitute the independent variable (xi, except 0 value) into the linear regression equation, and calculate the estimated value (y′) of the dependent variable (yi ), as well as the absolute and relative deviation between yi and y′, and the result should meet the requirements.
绝对偏差(mg/L)＝|yi-y'|………………………(6)Absolute deviation (mg/L)＝|yi -y'|……………………(6)
线性稀释倍数：Linear dilution factor:
(1)、线性实验要求：(1) Linear experiment requirements:
在[40，900]mg/L区间内，相关系数r应≥0.990。In the interval [40, 900] mg/L, the correlation coefficient r should be ≥ 0.990.
在[40，200)mg/L区间内，线性绝对偏差应不超过±20mg/L；Within the range of [40, 200) mg/L, the absolute deviation of linearity should not exceed ±20 mg/L;
在[200，900]mg/L区间内，线性相对偏差应不超过±10％。In the range of [200, 900]mg/L, the linear relative deviation should not exceed ±10%.
样本来源：合作医院(成都中医药大学附属医院)Sample source: Cooperative Hospital (Affiliated Hospital of Chengdu University of Traditional Chinese Medicine)
(2)、术语、定义、缩略词：(2), terms, definitions, acronyms:
试剂空白(R)：使用纯化水或指定空白液加入试剂作为样品测试，是判定试剂质量的一个有效指标。Reagent blank (R): Use purified water or a designated blank solution to add reagents as a sample test, which is an effective indicator for judging the quality of reagents.
定标反应度(S)：校准品标识值在反应体系中所测吸光度变化(ΔA)，是建立浓度与吸光度转化关系的重要指标。Calibration reaction degree (S): The change in absorbance (ΔA) measured by the identification value of the calibrator in the reaction system is an important indicator for establishing the conversion relationship between concentration and absorbance.
分析灵敏度：校准曲线的斜率。Analytical Sensitivity: The slope of the calibration curve.
线性:测量结果与样品中被测量的值直接成比例的能力。Linearity: The ability of a measurement result to be directly proportional to the value measured in the sample.
线性范围：测试系统可接收线性的范围，即在此范围内非线性误差小于规定误差。Linear range: The test system can accept the linear range, that is, the nonlinear error is less than the specified error within this range.
精密度：在规定条件下，对同一被测物进行连续多次测量所得结果之间的一致性。Precision: Under specified conditions, the consistency between the results of multiple consecutive measurements of the same measured object.
干扰:指在临床化学中，被测物浓度因样品特性或其他成分的影响而出现的临床显著性偏差。Interference: Refers to the clinically significant deviation of the concentration of the analyte due to the influence of sample characteristics or other components in clinical chemistry.
干扰标准：被测物浓度与真值间可产生的最大允许干扰偏差，此偏差可能影响医生的Interference standard: the maximum permissible interference deviation between the concentration of the measured substance and the true value, which may affect the doctor's
医疗决定。medical decisions.
抗干扰：确定干扰物质的干扰性。Anti-interference: Determine the interfering properties of interfering substances.
表1的试剂盒检测结果如下：The test results of the kit in Table 1 are as follows:
经实验发现，该测定试剂盒的相关系数r是符合规定的，但是不符合在40-200mg/L区间It is found through experiments that the correlation coefficient r of the assay kit is in compliance with the regulations, but not in the range of 40-200mg/L
内，线性绝对偏差应不超过±20mg/L；也不符合在200-900mg/L区间内，线性相对偏差应不超过±10％。低端偏差较大，线性较差，室间质评偏差大，无法达到准确检测的要求。Within the range of 200-900mg/L, the relative deviation of linearity should not exceed ±10%. The low-end deviation is large, the linearity is poor, and the external quality assessment deviation is large, which cannot meet the requirements of accurate detection.
实施例3色源物质浓度筛选Embodiment 3 color source substance concentration screening
1.实验方法：1. Experimental method:
根据表1的试剂盒组分和含量，配制含有不同色源物质的试剂，其中色源物质使用TOOS，浓度分别为：0.4mM、0.8mM、1.6mM、2.0mM、3.2mM。用不同TOOS浓度的试剂进行线性验证，线性做法及要求同上。According to the components and contents of the kit in Table 1, prepare reagents containing different color source substances, wherein TOOS is used as the color source material, and the concentrations are: 0.4mM, 0.8mM, 1.6mM, 2.0mM, 3.2mM. Use reagents with different TOOS concentrations for linearity verification, and the linearity method and requirements are the same as above.
2.实验结果：2. Experimental results:
表2色源物质浓度筛选Table 2 Concentration screening of color source substances
经实验发现，该测定试剂盒的相关系数r是符合规定的，但是从表2中，可以看出，表1试剂盒的试剂低端偏差较大，线性较差，本发明调整色源物质浓度，在浓度为0.4mM-3.2mM，均不符合在40-200mg/L区间内，线性绝对偏差应不超过±20mg/L；在浓度为0.4mM-3.2mM，不符合在200-900mg/L区间内，线性相对偏差应不超过±10％。由此可见，通过调整色源物质浓度仍不可对试剂盒试剂的线性进行优化。It is found through experiments that the correlation coefficient r of the assay kit meets the requirements, but from Table 2, it can be seen that the low-end deviation of the reagent kit in Table 1 is relatively large, and the linearity is poor. The present invention adjusts the concentration of the color source substance , when the concentration is 0.4mM-3.2mM, it does not conform to the range of 40-200mg/L, and the absolute deviation of linearity should not exceed ±20mg/L; Within the interval, the linear relative deviation should not exceed ±10%. It can be seen that the linearity of the kit reagents cannot be optimized by adjusting the concentration of the color source substance.
3.结论：3. Conclusion:
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整色源物质浓度仍不可对试剂盒试剂的线性进行优化。The linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents. The linearity of the reagents in the kit cannot be optimized by adjusting the concentration of the color source substance.
实施例4表面活性剂浓度筛选Embodiment 4 Surfactant concentration screening
1.实验目的：优化试剂线性1. The purpose of the experiment: to optimize the linearity of the reagent
2.实验方法：2. Experimental method:
根据表1的试剂盒组分和含量，配制含有不同表面活性剂的试剂，其中表面活性剂使用聚氧乙烯基联苯乙烯化苯基醚(A90)，浓度分别为：0.35g/L、0.7g/L、1.4g/L、1.75g/L、2.1g/L、2.8g/L。用不同A90浓度的试剂进行线性验证,线性验证方法同上。According to the kit components and contents in Table 1, prepare reagents containing different surfactants, wherein polyoxyethylene distyrenated phenyl ether (A90) is used as the surfactant, and the concentrations are: 0.35g/L, 0.7 g/L, 1.4g/L, 1.75g/L, 2.1g/L, 2.8g/L. Use reagents with different A90 concentrations to verify the linearity, and the linearity verification method is the same as above.
3.实验结果3. Experimental results
表3表面活性剂A90浓度筛选验证Table 3 Surfactant A90 concentration screening verification
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表3中，可以看出，调整表面活浓度，仍不能使试剂线性满足要求。It is found through experiments that the correlation coefficient r of the assay kit meets the requirements, but from Table 3, it can be seen that adjusting the surfactant concentration still cannot make the reagent linearly meet the requirements.
4.结论：4 Conclusion:
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整表面活性剂浓度仍不可对试剂盒试剂的线性进行优化。The linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of the surfactant.
实施例5底物4-氨基安替比林浓度的筛选The screening of embodiment 5 substrate 4-aminoantipyrine concentration
配制含有不同4-氨基安替比林浓度的试剂，4-氨基安替比林浓度为：2mM、4mM、8mM、12mM，用不同4-氨基安替比林浓度的试剂进行线性验证,线性验证方法同上。Prepare reagents containing different concentrations of 4-aminoantipyrine, the concentrations of 4-aminoantipyrine are: 2mM, 4mM, 8mM, 12mM, use reagents with different 4-aminoantipyrine concentrations for linearity verification, linearity verification The method is the same as above.
实验结果：Experimental results:
4-氨基安替比林浓度为2mM时试剂无反应度。其他4-氨基安替比林浓度的试剂验证结果如下：When the concentration of 4-aminoantipyrine is 2mM, there is no reagent reaction. The reagent verification results of other 4-aminoantipyrine concentrations are as follows:
表4 4-氨基安替比林浓度筛选验证Table 4 4-aminoantipyrine concentration screening verification
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表4中，可以看出，4-氨基安替比林浓度调高浓度后也并不能使试剂满足线性要求。It is found through experiments that the correlation coefficient r of the assay kit meets the requirements, but from Table 4, it can be seen that the reagent cannot meet the linearity requirement after the concentration of 4-aminoantipyrine is increased.
4.结论：4 Conclusion:
试剂盒试剂线性较差，不符合试剂的线性标准，通过调整4-氨基安替比林浓度仍不可对试剂盒试剂的线性进行优化。The linearity of the reagents in the kit is poor and does not meet the linearity standard of the reagents, and the linearity of the reagents in the kit cannot be optimized by adjusting the concentration of 4-aminoantipyrine.
实施例6曲拉通-100浓度筛选Example 6 Triton-100 Concentration Screening
配制含有不同曲拉通-100浓度的试剂，曲拉通-100的浓度为：5g/L、15g/L、25g/L、35g/L、50g/L。用不同曲拉通-100浓度的试剂进行线性验证,线性验证方法同上。Prepare reagents containing different concentrations of Triton-100, the concentrations of Triton-100 are: 5g/L, 15g/L, 25g/L, 35g/L, 50g/L. Reagents with different concentrations of Triton-100 were used for linearity verification, and the linearity verification method was the same as above.
表5曲拉通-100浓度筛选验证Table 5 Triton-100 Concentration Screening Validation
经实验发现，该测定试剂盒的相关系数r是符合规定的，但从表5中，可以看出，调整曲拉通-100的量也不能使试剂线性满足要求It is found through experiments that the correlation coefficient r of the assay kit meets the requirements, but from Table 5, it can be seen that adjusting the amount of Triton-100 cannot make the linearity of the reagent meet the requirements
4.结论：4 Conclusion:
现有技术中的试剂盒试剂线性较差，不符合试剂的线性标准，通过调整曲拉通-100浓度仍不可对试剂盒试剂的线性进行优化。The kit reagents in the prior art have poor linearity and do not meet the linearity standard of the reagents, and the linearity of the kit reagents cannot be optimized by adjusting the concentration of Triton-100.
实施例7用3-甲基苯并噻唑酮腙替换实施例1中的4-氨基安替比林的试剂Embodiment 7 replaces the reagent of 4-aminoantipyrine in embodiment 1 with 3-methylbenzothiazolone hydrazone
配制R2中含有3-甲基苯并噻唑酮腙，不含有4-氨基安替比林的试剂，3-甲基苯并噻唑酮腙浓度为0.4mol/L，其余组分与实施例1相同.Contain 3-methylbenzothiazolone hydrazone in the preparation R2, do not contain the reagent of 4-aminoantipyrine, the concentration of 3-methylbenzothiazolone hydrazone is 0.4mol/L, and the remaining components are the same as in Example 1 .
实验说明：Experiment description:
加速定标偏差计算公式式：(37℃定标反应度-4℃定标反应度)/4℃定标反应度。Accelerated calibration deviation calculation formula: (37°C calibration response -4°C calibration response)/4°C calibration response.
实验结果Experimental results
表6 4-氨基安替比林换为3-甲基苯并噻唑酮腙Table 6 4-aminoantipyrine is replaced by 3-methylbenzothiazolone hydrazone
经实验发现，该测定试剂盒的相关系数r是符合规定的，从表6中可以看出，配完立刻测定效果很好，线性满足要求，但试剂稳定性不满足要求。It is found through experiments that the correlation coefficient r of the assay kit meets the requirements. It can be seen from Table 6 that the assay effect is very good immediately after preparation, and the linearity meets the requirements, but the stability of the reagent does not meet the requirements.
4.结论4 Conclusion
本发明人意外的发现，将4-氨基安替比林替换为3-甲基苯并噻唑酮腙底物后试剂线性得到了优化，满足试剂线性标准的要求。The present inventors unexpectedly found that the linearity of the reagent is optimized after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone substrate, which meets the requirements of the linearity standard for the reagent.
实施例8试剂2防腐剂不同的试剂Different reagents of embodiment 8 reagent 2 preservatives
配制R2中含有3-甲基苯并噻唑酮腙，试用原缓冲类型(PIPES)，不同的防腐剂试剂，其中3-甲基苯并噻唑酮腙浓度为0.4mol/L，试剂分别为无防腐剂、叠氮钠1.0g/L、氟苯尼考1.0g/L、PC-300 1.0g/L。Contain 3-methylbenzothiazolone hydrazone in the preparation R2, try the original buffer type (PIPES), different preservative reagents, wherein the concentration of 3-methylbenzothiazolone hydrazone is 0.4mol/L, and the reagents are respectively non-preservative agent, sodium azide 1.0g/L, florfenicol 1.0g/L, PC-300 1.0g/L.
表7不同类型防腐剂验证Table 7 Verification of different types of preservatives
经实验发现，该测定试剂盒的相关系数r是符合规定的，从表7中可以看出：不同防腐剂的添加对R2稳定性有不同影响，不加防腐试剂稳定性最差，其次是叠氮钠和氟苯尼考。本发明人意外的发现，PC-300对于试剂稳定性验证中表现异常的好，说明PC-300不仅有防腐剂的作用，还具有明显提高R2试剂稳定性的作用。It is found through experiments that the correlation coefficient r of the assay kit meets the requirements. It can be seen from Table 7 that the addition of different preservatives has different effects on the stability of R2, and the stability without preservative reagents is the worst, followed by stacking Sodium Nitride and Florfenicol. The inventor unexpectedly found that PC-300 performed exceptionally well in reagent stability verification, indicating that PC-300 not only has the function of preservative, but also has the function of obviously improving the stability of R2 reagent.
实施例9试剂2缓冲类型不同的试剂Example 9 Reagents with different buffer types in reagent 2
配制R2中含有3-甲基苯并噻唑酮腙、PC-300，但缓冲类型不同的试剂，其中3-甲基苯 并噻唑酮腙浓度为0.4mol/L，不同类型的缓冲有：柠檬酸缓冲、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、硫酸、醋酸。Prepare reagents containing 3-methylbenzothiazolone hydrazone and PC-300 in R2, but with different buffer types, in which the concentration of 3-methylbenzothiazolone hydrazone is 0.4mol/L, and different types of buffers include: citric acid Buffer, Succinic Acid, Boric Acid, Tartaric Acid, Glycine, Hydrochloric Acid, Sulfuric Acid, Acetic Acid.
表8不同缓冲类型验证Table 8 Validation of different buffer types
经实验发现，该测定试剂的相关系数r是符合规定的，从表8和图1中可以看出：在pH值1.00-4.05的范围内，柠檬酸、丁二酸、硼酸、酒石酸、甘氨酸、盐酸、醋酸及其缓冲体系等酸性环境，与防腐剂PC-300产生协同作用，试剂稳定性显著得到改善，取得了预料不到的技术效果。It is found through experiments that the correlation coefficient r of the assay reagent is in compliance with the regulations, as can be seen from Table 8 and Figure 1: in the range of pH 1.00-4.05, citric acid, succinic acid, boric acid, tartaric acid, glycine, The acidic environment such as hydrochloric acid, acetic acid and its buffer system has a synergistic effect with the preservative PC-300, the stability of the reagent is significantly improved, and unexpected technical effects have been achieved.
总结：Summarize:
采用现有技术中4-氨基安替比林的试剂均不符合试剂的线性要求，即使通过调整色源物质浓度、表面活性剂浓度、4-氨基安替比林浓度、曲拉通-100浓度后均不可对线性进行优化，线性均无法满足试剂准确性的要求。本发明将4-氨基安替比林替换为3-甲基苯并噻唑酮腙后试剂的线性得到了改善，满足试剂准确性的要求，但是发明人发现体系的稳定性不高。通过实验意外发现，选择PC-300作为防腐剂，兼具了稳定试剂体系的作用，已经满足了性能优化的要求。进一步，通过将pH值调整至1.00-4.05范围，能与PC-300产生协同作用，极大提高了体系的稳定性，取得了预料不到的技术效果。The reagents using 4-aminoantipyrine in the prior art all do not meet the linearity requirements of the reagents, even by adjusting the concentration of the color source substance, the concentration of the surfactant, the concentration of 4-aminoantipyrine, and the concentration of Triton-100 After that, the linearity cannot be optimized, and the linearity cannot meet the requirements of reagent accuracy. In the present invention, the linearity of the reagent is improved after replacing 4-aminoantipyrine with 3-methylbenzothiazolone hydrazone, which meets the requirement of reagent accuracy, but the inventor finds that the stability of the system is not high. It was unexpectedly discovered through experiments that PC-300 was selected as a preservative, which has the function of stabilizing the reagent system and has met the requirements of performance optimization. Further, by adjusting the pH value to the range of 1.00-4.05, it can produce a synergistic effect with PC-300, greatly improve the stability of the system, and achieve unexpected technical effects.
实施例10试剂线性验证Example 10 Reagent Linearity Verification
按照表9组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表9试剂在全自动生化分析仪中进行测定。测定结果如表10所示。Prepare detection reagents according to the components in Table 9, use the method in Example 2 to prepare samples with gradient concentrations, and use the reagents in Table 9 to perform determination in an automatic biochemical analyzer. The measurement results are shown in Table 10.
表9试剂组分Table 9 Reagent Components
表10线性验证Table 10 Linearity Verification
从表10可以看出，使用本发明的3-甲基苯并噻唑酮测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差均非常小，完全满足线性要求。As can be seen from Table 10, the correlation coefficient r using the 3-methylbenzothiazolone assay reagent of the present invention is in compliance with the regulations, and the absolute deviation and relative deviation in the low value area are very small, fully meeting the linearity requirement.
实施例11试剂线性验证Example 11 Reagent Linearity Verification
按照表11组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表11试剂在全自动生化分析仪中进行测定。测定结果如表12所示。Prepare detection reagents according to the components in Table 11, use the method in Example 2 to prepare samples with gradient concentrations, and use the reagents in Table 11 for determination in an automatic biochemical analyzer. The measurement results are shown in Table 12.
表11试剂组分Table 11 Reagent Components
表12线性验证Table 12 Linearity Verification
从表12可以看出，该测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差也非常小，完全满足线性要求。It can be seen from Table 12 that the correlation coefficient r of the assay reagent is in compliance with the regulations, and the absolute deviation and relative deviation in the low value area are also very small, fully meeting the linearity requirement.
实施例12试剂线性验证Example 12 Reagent Linearity Verification
按照表13组分配制检测试剂，用实施例2的方法配制梯度浓度的样品，用表11试剂在全自动生化分析仪中进行测定。测定结果如表14所示。Prepare detection reagents according to the components in Table 13, use the method in Example 2 to prepare samples with gradient concentrations, and use the reagents in Table 11 to perform determination in an automatic biochemical analyzer. The measurement results are shown in Table 14.
表13试剂组分Table 13 Reagent Components
表14线性验证Table 14 Linearity Verification
从表14可以看出，该测定试剂的相关系数r是符合规定的，低值区域绝对偏差和相对偏差也非常小，完全满足线性要求。It can be seen from Table 14 that the correlation coefficient r of the assay reagent is in compliance with the regulations, and the absolute deviation and relative deviation in the low value area are also very small, fully meeting the linearity requirement.
实施例13本申请的试剂与市售产品相关性验证Embodiment 13 Reagent of the present application and commercially available product correlation verification
分别采用本发明实施例10、11和12的试剂与市售试剂(九强20-0630)，使用实施例2的方法，采用日立7180全自动生化分析仪，对100份样本，按各自参数进行测定，并对测定值进行相关分析(横纵坐标单位mg/L)。Respectively adopt the reagent of the embodiment of the present invention 10,11 and 12 and commercially available reagent (Jiuqiang 20-0630), use the method for embodiment 2, adopt Hitachi 7180 automatic biochemical analyzer, to 100 samples, carry out by respective parameter Determination and correlation analysis of the measured values (abscissa and ordinate unit mg/L).
结果如图2-4所示，本发明的试剂与市售试剂相比，一致性程度高。The results are shown in Figures 2-4, compared with commercially available reagents, the reagent of the present invention has a higher degree of consistency.
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
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| JP2016034252A (en)* | 2014-08-04 | 2016-03-17 | 東洋紡株式会社 | Method and reagent for stabilizing cholesterol esterase |
| CN107446989A (en)* | 2017-06-23 | 2017-12-08 | 美康生物科技股份有限公司 | Determine the method and reagent of the sdLDL-C in sample |
| CN112074743A (en)* | 2018-03-28 | 2020-12-11 | 电化株式会社 | Method, reagent and kit for quantifying lipoprotein cholesterol |
| CN114214389A (en)* | 2022-02-21 | 2022-03-22 | 北京沃森赛瑟生物技术有限公司 | Lipoprotein cholesterol detection method and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114214389B (en) | 2022-07-12 |
| CN114214389A (en) | 2022-03-22 |
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